This renewal proposal explores mechanisms that regulate alternative pre-mRNA splicing in differentiating erythroid cells, focusing on cis-regulatory sequences and trans-acting splicing factor proteins that regulate erythroid stage-specific switches in exon splicing. Studies will investigate the prototypical splicing switch in late erythroblasts that activates protein 4.1R exon 16 (E16), known to be physiologically important for mechanical stabilization of the red cell membrane, and several newly appreciated and evolutionarily conserved splicing switches in other erythroid pre-mRNAs. Major aims of the proposal include (1) affinity purification of novel factors that antagonize or synergize with Fox2 enhancer function in the highly conserved intron downstream of E16;(2) testing the hypothesis that four new erythroid splicing switches are coordinately regulated with E16, by Fox2 or other E16-regulatory factors, to provide new insights into the broader erythroid splicing program;and (3) initiation of a new effort to characterize functionality of cis-regulatory elements and trans-splicing factors in vivo with animal models. In addition to its biological importance for erythroid function, the E16 splicing switch is one of the best models for analysis of tissue-specific splicing in any cell system. Innovative features of the proposed studies include: detailed analysis of the conserved intron flanking the intron 16 Fox2 sites, that may provide insight into modulation of Fox-2 splicing activity;initial studies of the putatively co-regulated splicing switches in several other erythroid pre-mRNAs;and in vivo analysis of E16 regulatory elements. Successful accomplishment of these objectives will lead to a better understanding of the stage-specific switch in 4.1R premRNA splicing, and may provide preliminary evidence regarding a potential larger role for Fox-2 in mediating the erythroid differentiation stage-specific alternative splicing program. These studies should also provide insights into disease mechanisms caused by aberrant splicing, ultimately leading to splicing therapeutics to correct such defects.